Protein Isolation
Protein isolation is an important tool in biological research, clinical diagnostics and the production of pharmaceuticals, especially production by recombinant techniques. The scientific researcher must obtain a protein quickly while retaining high specific activity; the clinician must identify proteins in biological samples in order to make an accurate diagnosis; and the molecular biologist must recover and purify large quantities of proteins produced by recombinant organisms.
Scientists have traditionally isolated proteins by precipitating them from biological samples with salts, such as ammonium sulfate, or organic solvents, such as ethanol. Exposure to the chemicals used in such methods often causes protein denaturation. In addition, separation of the proteins from the precipitating chemical is difficult and may cause further denaturation.
The ammonium sulfate precipitation technique, also known as "salting out," is based on the fact that the solubility of most proteins decreases at high electrolyte concentration. Sulfate is used because multivalent ions are more effective than monovalent ions. This procedure is usually carried out in the cold (0.degree.-4.degree. C.) with control of pH close to neutrality. Different classes of proteins precipitate depending on the concentration of salt added. The disadvantage to this method is the difficulty of removing residual salt from the precipitate or supernatant. Often dialysis is used, but is very time consuming.
Organic solvents are often used for fractional precipitation of proteins. However, there is a risk that the solvent will denature the protein unless kept at a temperature near the freezing point. In addition, the solvent must be removed from the protein. A solvent such as ethanol is generally removed by lyophilizing the precipitated proteins.
Recent advances in protein purification have centered around the development of high performance ion exchange, affinity chromatography, hydrophobic interactions and gel filtration chromatography. The biological sample is loaded onto a chromatography column and is eluted with the appropriate solvent into fractions that are analyzed for protein activity. This method is expensive, time consuming, and poorly suited for large scale protein purification.
Many scientist continue to use traditional methods alone or in combination with the recently developed chromatography procedures. For example, after precipitation of a protein from a biological sample with ammonium sulfate, protein is separated from the ammonium sulfate salt by chromatography. Often the protein loses activity or becomes denatured during one or more steps of the procedure, resulting in a low yield or inaccurate identification.
Polymer Studies
In the mid 1960's, Polson et al., Biochim. Biophys. Acta 82:463-475 (1964), analyzed a variety of high molecular weight polymers for purifying proteins including polyethylene glycol (PEG), dextran, nonylphenol-ethoxylates (NPEs), polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP). Polson et al. concluded that PEG is the most suitable of the tested polymers for protein precipitation. The disadvantage to PEG is that it must be removed from the protein either by passage through a DEAE or cellulose column under conditions that adsorb the proteins and wash out the PEG with the effluent or by adding ethanol to precipitate the proteins from the supernatant, leaving the PEG in the supernatant. Polson et al. explicitly caution against the use of PVE, PVP and NPEs because of their high intrinsic viscosities and because, according to their observations, the polymers cause significant protein denaturation. The results of Polson et al. were confirmed and extended by Zeppezauer and Brishammar, Biochim. Biophys. Acta 94:581-583 (1965) who precipitated kidney proteins with three high molecular weight preparations of Polyox.TM. a PEG resin obtained from Union Carbide Corp. (Danbury, Conn.).
Polyethylene glycol (PEG), also known as poly(oxyethylene)glycol, is a condensation polymer of ethylene oxide and water having the general chemical formula HO(CH.sub.2 CH.sub.2 O).sub.n H. PEG is used as a water-soluble lubricant for rubber, textile and metal manufacture; in food, cosmetics, water paints, paper coatings, and polishes; and as an ointment base in pharmaceuticals.
Dextran is a term applied to polysaccharides produced by bacteria growing on a sucrose substrate. Native dextrans produced by bacteria such as Leuconostoc mesenteroides and Lactobacteria dextranicum usually have a high molecular weight. The lower molecular weight dextrans used as plasma volume expanders or blood flow adjuvants are usually prepared by depolymerization of native dextrans or by synthesis.
NPEs are a class of long chained compounds often used as surfactants. They are usually derivatized to meet the desired solubility requirements.
PVA is a polymer prepared from polyvinyl acetates by replacement of the acetate groups with hydroxyl groups and has the formula (CH.sub.2 CHOH).sub.n. Most polyvinyl alcohols are soluble in water and are used as elastomers in the plastics industry, as viscosity increasing agents in the pharmaceutical industry, and as ophthalmic lubricants.
PVP is a non-ionogenic, hydrophilic polymer having a mean molecular weight ranging from approximately 10,000 to 700,000 and the chemical formula (C.sub.6 H.sub.9 NO).sub.n. PVP is also known as poly[1-(2-oxo-1-pyrrolidinyl)ethylene], Povidone.TM., Polyvidone.TM., RP 143.TM., Kollidon.TM., Peregal ST.TM., Periston.TM., Plasdone.TM., Plasmosan.TM., Protagent.TM., Subtosan.TM., and Vinisil.TM.. PVP is non-toxic, highly hygroscopic and readily dissolves in water or organic solvents. PVP has a wide variety of uses such as in pharmaceuticals, as a complexing agent, and for the detoxification of chemicals. It is also used in tableting, photographic emulsions, cosmetics, detergents, adhesives, and beer and wine clarification. PVP was used as a blood plasma expander during World War II, but when high molecular weights were found to be adsorbed in tissues this use was abandoned. intravenous PVP has been used to decrease human serum lipids as described by Sanbar and Smet, Circulation 38:771-776 (1968).
PEG, dextran, PVA and PVP are commercially available from chemical suppliers such as the Sigma Chemical Company (St. Louis, Mo.). NPEs require custom synthesis and can be ordered from special chemical producers.
Recombinant Techniques
Many proteins, such as for example, human growth hormone and insulin, are now produced by recombinant techniques. The gene encoding the protein is inserted into a bacterial or viral vector causing production of large quantities of the protein which must then be isolated from the other proteins in the growth media, or fermentation fluid. A rapid, inexpensive method for the purification of proteins produced by recombinant techniques would help reduce the costs and improve the recovery of proteins produced in this manner.
Drug Disposition
A sufficient amount of pharmaceutical agent or drug must reach its site of action in order to exert a desired effect. Drug absorbed into the blood from the site of administration often binds to proteins, such as albumin, that retard the delivery of drug to the site of action. A drug having a higher affinity for serum proteins will require a larger dose to achieve the desired effect.
During pharmaceutical development, drug disposition studies are performed to determine the amount of drug bound to serum protein. After administration of drug, serum proteins are isolated by chromatography or are precipitated by chemicals such as ammonium sulfate. The concentration of the drug in the protein fraction is determined and is compared with the total concentration of drug found in the intact sample through analytical techniques. These methods are time consuming and do not provide sufficient information concerning the identity of the proteins to which the drug is bound.
Urine samples are also analyzed for drug or drug metabolite concentration to ensure that the drug is excreted and is not retained by the body. Interfering proteins are often separated from drug as described above using time consuming procedures.
Albumin Isolation
Albumin is a simple protein distributed throughout the tissues and fluids of plants and animals, well known for its presence in the white portion of poultry eggs. Albumin is soluble in water and is easily denatured by heat, acid or neutral solutions. Bovine serum albumin (BSA) is derived from bovine blood and is often used in in vitro biological studies. Normal human serum albumin is obtained by fractionating blood plasma proteins from healthy persons and is used as a transfusion material. Serum albumin is also used in diagnostics such as, for example, the use of radioiodinated serum albumin in determining blood volume and cardiac output. Therefore, there is a great need for an inexpensive method of producing large quantities of purified albumin.
Immunoglobulin Isolation
The immunoglobulins IgG, IgM, IgA, IgE and IgD, which are found in the gamma globulin fraction of vertebrate serum proteins, constitute the circulating antibody population and provide the humoral immune response necessary to fight infection and disease. A measurement of the serum globulin to albumin ratio provides a good indication of the presence of an immune response to infection and an individual's ability to combat the infection. An abnormally high concentration of globulin in the serum is often an indication of a hyperproliferative disorder such as myeloma or Bence Jones proteins. Purified immunoglobulins are necessary for scientific research, especially in the development of vaccines, and for passive immunization of individuals who have been recently exposed to a bacteria or virus for which a vaccine is not yet available. Therefore, a rapid method for isolating immunoglobulins from blood for research, diagnostic or therapeutic purposes is necessary.
Antibodies
Monoclonal antibodies are created by fusing a normal antibody-producing lymphocyte from the spleen of a recently immunized experimental animal to a myeloma cell line to form a hybridoma. The myeloma cell causes the continuous production of the antibody of interest which is usually recovered from ascites fluid. Monoclonal antibodies must be isolated from the other proteins present in the ascites fluid before use as reagents in diagnostic kits, scientific research, or coupled to a drug to provide a "magic bullet" that is directed to a target site such as a malignant tumor. Polyclonal antibodies are produced by injecting an animal, such as a mouse, rat or rabbit, with an antigen, collecting blood, and isolating the immunoglobulin fraction that binds to the antigen, usually by passage of the immunoglobulin fraction through an affinity column to which antigen has been immobilized. The resulting polyclonal antibodies are used for the same purposes as monoclonal antibodies described above except that the specificity of a polyclonal antibody for a particular antigen is not as great. An inexpensive, rapid method of isolating and purifying monoclonal or polyclonal antibodies would greatly simplify antibody production.
Spinal Fluid and Urine Analysis
Medical diagnosis of disease or disorders is often achieved by analyzing bodily fluids such as spinal fluid or urine. Separation of biomolecules from interfering substances in the spinal fluid or urine sample would provide a faster, more reliable diagnosis.
What is needed is a biomolecule isolation method that is simple, inexpensive and fast, yet allows for the isolation of a relatively pure, active biomolecule.
It is therefore an object of the present invention to provide a non-denaturing method of isolating a biomolecule.
It is a further object of the present invention to provide a rapid, reproducible method of isolating a relatively pure protein.
It is a further object of the present invention to provide a method of isolating a biomolecule from a biological sample in a single step.
It is a further object of the present invention to provide a method of isolating large quantities of relatively pure protein.
It is a further object of the present invention to provide a method of determining the globulin to albumin ratio in serum.
It is a further object of the present invention to provide a method of determining the disposition of a drug in serum proteins.